Ligand functionalized polymers

ABSTRACT

Ligand functionalized substrates, methods of making ligand functionalized substrates, and methods of using functionalized substrates are disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/305,740, filed Feb. 18, 2010, the disclosure of whichis incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present disclosure relates to ligand-functionalized polymers, andmethods for preparing the same. The functionalized polymers are usefulin selectively binding and removing biological materials, such asviruses, from biological samples.

BACKGROUND

Detection, quantification, isolation and purification of targetbiomaterials, such as viruses and biomacromolecules (includingconstituents or products of living cells, for example, proteins,carbohydrates, lipids, and nucleic acids) have long been objectives ofinvestigators. Detection and quantification are importantdiagnostically, for example, as indicators of various physiologicalconditions such as diseases. Isolation and purification ofbiomacromolecules are important for therapeutic uses and in biomedicalresearch. Biomacromolecules such as enzymes which are a special class ofproteins capable of catalyzing chemical reactions are also usefulindustrially; enzymes have been isolated, purified, and then utilizedfor the production of sweeteners, antibiotics, and a variety of organiccompounds such as ethanol, acetic acid, lysine, aspartic acid, andbiologically useful products such as antibodies and steroids.

In their native state in vivo, structures and corresponding biologicalactivities of these biomacromolecules are maintained generally withinfairly narrow ranges of pH and ionic strength. Consequently, anyseparation and purification operation must take such factors intoaccount in order for the resultant, processed biomacromolecule to havepotency.

The use of certain ionic polymers, especially cationic polymers, for theflocculation of cell and/or cell debris, as well as for theprecipitation of proteins, is known. Similarly, ionic polymers have beenused to modify filtration media to enhance the removal of impuritiesfrom process streams in depth filtration or membrane absorber typeapplications. The effectiveness of these flocculants is typicallyreduced as the conductivity of the media being processed increases, i.e.as the salt content increases. There is a need in the art for polymericmaterials with increased affinity for biological species under highionic strength conditions.

Chromatographic separation and purification operations can be performedon biological product mixtures, based on the interchange of a solutebetween a moving phase, which can be a gas or liquid, and a stationaryphase. Separation of various solutes of the solution mixture isaccomplished because of varying binding interactions of each solute withthe stationary phase; stronger binding interactions generally result inlonger retention times when subjected to the dissociation ordisplacement effects of a mobile phase compared to solutes whichinteract less strongly and, in this fashion, separation and purificationcan be effected.

Most current capture or purification chromatography is done viaconventional column techniques. These techniques have severebottlenecking issues in downstream purification, as the throughput usingthis technology is low. Attempts to alleviate these issues includeincreasing the diameter of the chromatography column, but this in turncreates challenges due to difficulties of packing the columnseffectively and reproducibly. Larger column diameters also increase theoccurrence of problematic channeling. Also, in a conventionalchromatographic column, the absorption operation is shut down when abreakthrough of the desired product above a specific level is detected.This causes the dynamic or effective capacity of the adsorption media tobe significantly less than the overall or static capacity. Thisreduction in effectiveness has severe economic consequences, given thehigh cost of some chromatographic resins.

Polymeric resins are widely used for the separation and purification ofvarious target compounds. For example, polymeric resins can be used topurify or separate a target compound based on the presence of an ionicgroup, based on the size of the target compound, based on a hydrophobicinteraction, based on an affinity interaction, or based on the formationof a covalent bond. There is a need in the art for polymeric substrateshaving enhanced affinity for viruses to allow selective removal from abiological sample. There is further need in the art for ligandfunctionalized membranes that overcome limitations in diffusion andbinding, and that may be operated at high throughput and at lowerpressure drops.

SUMMARY OF THE INVENTION

The present invention is directed to ligand-functionalized polymers, andmethods of making the same. More specifically, the ligand-functionalizedpolymer includes a base polymer, having carbonyl functional groups,which has been modified to provide grafted ligand groups having therequisite affinity for binding neutral or negatively chargedbiomaterials, such as cells, cell debris, bacteria, spores, viruses,nucleic acids, and proteins.

In some embodiments, the ligand-functionalized polymer may be used as aflocculant whereby a biological sample, such as a cell culture fluid, iscontacted causing negative and/or neutral species to bind to the polymerand precipitate from the solution or suspension. In another embodiment,a base substrate, such as a microporous membrane or a particle, may becoated with the ligand-functionalized polymer. In another embodiment,the ligand-functionalized polymer may be grafted to the surface of abase substrate.

The ligand functionalized polymer may be described as the reactionproduct of a carbonyl-functional polymer, such as diacetone(meth)acrylate (co)polymer, and a ligand compound of Formula I:

whereinR² is a covalent bond, a C₂ to C₁₂ alkylene, a C₅-C₁₂ (hetero)arylene,

R⁹ is C₂ to C₁₂ alkylene or C₅-C₁₂ (hetero)arylene,each R³ is independently H, —OH, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,preferably H or C₁-C₄ alkyl,R⁴ is H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or —N(R³)₂, preferably H, orC₁-C₄ alkyl.

Methods of making a ligand functionalized substrate are provided. Insome embodiments, the method comprises reacting a carbonyl-functionalpolymer with the ligand compound of Formula I, optionally in thepresence of an acid catalyst.

A functionalized polymer is provided having grafted pendent ligandgroups, said ligand groups of Formula II:

whereinR¹ is H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl, preferably C₁-C₁₂ alkyl;R² is a covalent bond, a C₂ to C₁₂ alkylene, or a C₅-C₁₂(hetero)arylene,

R⁹ is C₂ to C₁₂ alkylene or C₅-C₁₂ (hetero)arylene,each R³ is independently H, —OH, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,preferably H or C₁-C₄ alkyl,R⁴ is H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or —N(R³)₂, preferably H, orC₁-C₄ alkyl.

It will be recognized that the

group of Formula II is the linkage formed between the terminal amine ofthe ligand compound of Formula I and the carbonyl group of thecarbonyl-functional polymer. With respect to the above Formula II, the“˜” represents a covalent bond or an organic linking group interposedbetween the ligand group and polymer chain.

In other embodiments, ligand functional polymer may be prepared in whichthe imine linking group (˜˜C(R¹)═N— is reduced to an amine linking group(˜˜CH(R¹)—NH—. This may be effected by treating the extant ligandfunctional polymer with a reducing agent, such as sodiumcyanoborohydride, or the reduction may be effected in situ by added thereducing agent to the reaction mixture of the carbonyl functionalpolymer and the compound of Formula I.

In this application, (meth)acrylic is inclusive of both methacrylic andacrylic.

As used herein, “alkyl” or “alkylene” includes straight-chained,branched, and cyclic alkyl groups and includes both unsubstituted andsubstituted alkyl groups. Unless otherwise indicated, the alkyl groupstypically contain from 1 to 20 carbon atoms. Examples of “alkyl” as usedherein include, but are not limited to, methyl, ethyl, n-propyl,n-butyl, n-pentyl, isobutyl, t-butyl, isopropyl, n-octyl, n-heptyl,ethylhexyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, andnorbornyl, and the like. Unless otherwise noted, alkyl groups may bemono- or polyvalent.

As used herein, “aryl” or “arylene” is an aromatic group containing 5-12ring atoms and can contain optional fused rings, which may be saturated,unsaturated, or aromatic. Examples of an aryl groups include phenyl,naphthyl, biphenyl, phenanthryl, and anthracyl. Heteroaryl is arylcontaining 1-3 heteroatoms such as nitrogen, oxygen, or sulfur and cancontain fused rings. Some examples of heteroaryl groups are pyridyl,furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,benzofuranyl, and benzthiazolyl. Unless otherwise noted, aryl andheteroaryl groups may be mono- or polyvalent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the bovine serum albumin precipitation data ofExample 1-5.

DETAILED DESCRIPTION OF THE INVENTION

In the article and methods of this invention, ligand-functionalizedpolymers are provided which have enhanced affinity, especially in highionic strength media, for neutral or negatively charged biologicalmaterials such as host cell proteins, DNA, RNA, viruses, and othermicroorganisms. The affinity for such biomaterials allows positivelycharged materials, such as antibodies, to be purified, as they are notbound to the ligand functional groups. The ligand functionalizedsubstrate allows the selective capture or binding of target biomaterialsby the ligand groups, while other materials, lacking the affinity forthe ligand groups are passed. In some embodiments the ligandfunctionalized polymer is used as a flocculant to selectively bindtarget biomaterials, precipitate them from solution, and theprecipitated adduct subsequently separated.

The base polymer comprises a carbonyl-functional (co)polymer; i.e. apolymer having aldehyde or ketone groups, typically pendent from thepolymer chain. The polymers comprise polymerized monomer units of anethylenically unsaturated monomer having a carbonyl group, preferably aketone group, or copolymerized monomer units of an ethylenicallyunsaturated monomer and carbon monoxide (Carbon monoxide copolymers).

Generally, the carbonyl-functional (co)polymer is selected from thegroup consisting of; carbon monoxide, acrolein, vinyl methyl ketone,vinyl ethyl ketone, vinyl isobutyl ketone, isopropenyl methyl ketone,vinyl phenyl ketone, diacetone (meth)acrylamide, acetonyl acrylate,acetoacetoxyethyl (meth)acrylate, and diacetone (meth)acrylate(co)polymers. An example of a carbon monoxide containing copolymer isELVALOY™ 741, a terpolymer of ethylene/vinyl acetate/carbon monoxide.

The polymer may be a copolymer of the carbonyl-functional monomer units.In particular, the carbonyl functional polymer may further compriseethylenically unsaturated hydrophilic monomer units, and/or hydrophobicmonomer units. The copolymer, in some embodiments, may be a crosslinkedcopolymer. In particular, the carbonyl functional copolymer may furthercomprise comonomer units having more than one ethylenically unsaturatedgroups.

As used herein “hydrophilic monomers” are those polymerizable monomershaving a water miscibility (water in monomer) of at least 1 wt. %,preferably at least 5 weight % without reaching a cloud point, andcontain no functional groups that would interfere with the binding ofbiological substances to the ligand group. The copolymer may comprise 0to 90 wt. % of such monomer units in the monomer solution. When present,the polymer generally comprises 1 to 90 wt. % of such of such monomerunits based on 100 wt. % total monomer.

The hydrophilic groups of the hydrophilic monomers may be neutral, havea positive charge, a negative charge, or a combination thereof. Withsome suitable ionic monomers, the ionic group can be neutral or chargeddepending on the pH conditions. This class of monomers is typically usedto impart a desired hydrophilicity, i.e. water solubility ordispersibility to the copolymer. These comonomers are typically used toimpart a desired water solubility/dispersibility of the ligandfunctionalized copolymer. A negatively charged comonomer may be includedas long as it is in small enough amounts that it doesn't interfere withthe ligand binding interaction. In applications for viral capture, theaddition of a hydrophilic monomer having a positive charge at theselected pH may allow selective binding and flocculation of viruseswhile repelling positively charged biological materials such asantibodies.

Some exemplary ionic monomers that are capable of providing a positivecharge are amino (meth)acrylates or amino (meth)acrylamides of FormulaIV or quaternary ammonium salts thereof. The counter ions of thequaternary ammonium salts are often halides, sulfates, phosphates,nitrates, and the like.

where X is —O— or —NR³—;R⁷ is independently H or CH₃,R⁶ is a C₂ to C₁₀ alkylene, preferably C₂-C₆.Each R⁸ is independently hydrogen, alkyl, hydroxyalkyl (i.e. an alkylsubstituted with a hydroxy), or aminoalkyl (i.e. an alkyl substitutedwith an amino). Alternatively, the two R⁸ groups taken together with thenitrogen atom to which they are attached can form a heterocyclic groupthat is aromatic, partially unsaturated (i.e. unsaturated but notaromatic), or saturated, wherein the heterocyclic group can optionallybe fused to a second ring that is aromatic (e.g. benzene), partiallyunsaturated (e.g. cyclohexene), or saturated (e.g. cyclohexane).

It will be understood with respect to Formula IV that the depicted(meth)acrylate group may be replaced by another ethylenicallyunsaturated group of reduced reactivity, such as methacrylate,(meth)acrylamide, vinyl, vinyloxy, allyl, allyloxy, and acetylenyl.

In some embodiments of Formula IV, both R⁸ groups are hydrogen. In otherembodiments, one R⁸ group is hydrogen and the other is an alkyl having 1to 10, 1 to 6, or 1 to 4 carbon atoms. In still other embodiments, atleast one of R⁸ groups is a hydroxy alkyl or an amino alkyl that have 1to 10, 1 to 6, or 1 to 4 carbon atoms with the hydroxy or amino groupbeing positioned on any of the carbon atoms of the alkyl group. In yetother embodiments, the R⁸ groups combine with the nitrogen atom to whichthey are attached to form a heterocyclic group. The heterocyclic groupincludes at least one nitrogen atom and can contain other heteroatomssuch as oxygen or sulfur. Exemplary heterocyclic groups include, but arenot limited to imidazolyl. The heterocyclic group can be fused to anadditional ring such as a benzene, cyclohexene, or cyclohexane.Exemplary heterocyclic groups fused to an additional ring include, butare not limited to, benzoimidazolyl.

Exemplary amino acrylates (i.e. X in Formula IV is oxy) includeN,N-dialkylaminoalkyl (meth)acrylates such as, for example,N,N-dimethylaminoethyl(meth)acrylate, N,N-dimethylaminoethylacrylate,N,N-diethylaminoethylacrylate, N,N-dimethylaminopropyl(meth)acrylate,N-tert-butylaminopropyl(meth)acrylate, and the like.

Exemplary amino (meth)acrylamides (i.e. X in Formula IV is —NR³—)include, for example, N-(3-aminopropyl)methacrylamide,N-(3-aminopropyl)acrylamide, N-[3-(dimethylamino)propyl]methacrylamide,N-[3-(dimethylamino)propyl]acrylamide,N-(3-imidazolylpropyl)methacrylamide, N-(3-imidazolylpropyl)acrylamide,N-(2-imidazolylethyl)methacrylamide,N-(1,1-dimethyl-3-imidazolylpropyl)methacrylamide,N-(1,1-dimethyl-3-imidazolylpropyl)acrylamide,N-(3-benzimidazolylpropyl)acrylamide, andN-(3-benzimidazolylpropyl)methacrylamide.

Exemplary quaternary salts of the monomers of Formula IV include, butare not limited to, (meth)acrylamidoalkyltrimethylammonium salts (e.g.3-methacrylamidopropyltrimethylammonium chloride and3-acrylamidopropyltrimethylammonium chloride) and(meth)acryloxyalkyltrimethylammonium salts (e.g.2-acryloxyethyltrimethylammonium chloride,2-methacryloxyethyltrimethylammonium chloride,3-methacryloxy-2-hydroxypropyltrimethylammonium chloride,3-acryloxy-2-hydroxypropyltrimethylammonium chloride, and2-acryloxyethyltrimethylammonium methyl sulfate).

Other monomers that can provide positively charged groups to the polymerinclude the dialkylaminoalkylamine adducts of alkenylazlactones (e.g.2-(diethylamino)ethylamine, (2-aminoethyl)trimethylammonium chloride,and 3-(dimethylamino)propylamine adducts of vinyldimethylazlactone) anddiallylamine monomers (e.g. diallylammonium chloride anddiallyldimethylammonium chloride).

In some preferred embodiments, the second hydrophilic monomer may havean acrylate group, or other ethylenically unsaturated groups, and apoly(alkylene oxide) group; e.g. monoacrylated poly(alkylene oxidecompounds, where the terminus is a hydroxy group, or an alkyl ethergroup. Such monomers are of the general formula:R³—O—(CH(R³)—CH₂—O)_(n)—C(O)—C(R³)═CH₂,  V,wherein each R³ is independently H or C₁-C₄ alkyl, and n is at least 2.

In one embodiment, the poly(alkylene oxide) group (depicted as—(CH(R³)—CH₂—O)_(n)—) is a poly(ethylene oxide) (co)polymer. In anotherembodiment, the poly(alkylene oxide) group is a poly(ethyleneoxide-co-propylene oxide) copolymer. Such copolymers may be blockcopolymers, random copolymers, or gradient copolymers.

Other representative examples of suitable hydrophilic monomers includebut are not limited to acrylic acid; methacrylic acid;2-acrylamido-2-methyl-1-propanesulfonic acid; 2-hydroxyethyl(meth)acrylate; N-vinylpyrrolidone; N-vinylcaprolactam; acrylamide;mono- or di-N-alkyl substituted acrylamide; t-butyl acrylamide;dimethylacrylamide; N-octyl acrylamide; poly(alkoxyalkyl)(meth)acrylates including 2-(2-ethoxyethoxy)ethyl (meth)acrylate,2-ethoxyethyl (meth)acrylate, 2-methoxyethoxyethyl (meth)acrylate,2-methoxyethyl methacrylate, polyethylene glycol mono(meth)acrylates;alkyl vinyl ethers, including vinyl methyl ether; and mixtures thereof.Preferred hydrophilic monomers include those selected from the groupconsisting of dimethylacrylamide, 2-hydroxyethyl (meth)acrylate andN-vinylpyrrolidinone.

The copolymer may further comprise hydrophobic monomer units, in amountsthat do not deleteriously affect the binding performance of the ligandpolymer, and the water dispersibility thereof. When present, the polymergenerally comprises 1 to 20 wt. % of such monomer units based on 100 wt.% total monomer.

Useful classes of hydrophobic monomers include alkyl acrylate esters andamides, exemplified by straight-chain, cyclic, and branched-chainisomers of alkyl esters containing C₁-C₃₀ alkyl groups and mono- ordialkyl acrylamides containing C₁-C₃₀ alkyl groups. Useful specificexamples of alkyl acrylate esters include: methyl acrylate, ethylacrylate, n-propyl acrylate, n-butyl acrylate, iso-amyl acrylate,n-hexyl acrylate, n-heptyl acrylate, isobornyl acrylate, n-octylacrylate, iso-octyl acrylate, 2-ethylhexyl acrylate, iso-nonyl acrylate,decyl acrylate, undecyl acrylate, dodecyl acrylate, lauryl acrylate,tridecyl acrylate, and tetradecyl acrylate. Useful specific examples ofalkyl acrylamides include mono- and diacrylamides having pentyl, hexyl,heptyl, isobornyl, octyl, 2-ethylhexyl, iso-nonyl, decyl, undecyl,dodecyl, tridecyl, and tetradecyl groups may be used. The correspondingmethacrylate esters may be used.

Useful classes of hydrophobic monomers further include vinyl monomerssuch as vinyl acetate, styrenes, and alkyl vinyl ethers, maleicanhydride and polyfunctional monomers.

The ligand functional polymer may be prepared by condensation of thecarbonyl functional (co)polymer with a ligand compound of Formula I:

WhereinR² is a covalent bond, a C₂ to C₁₂ alkylene, a C₅-C₁₂ (hetero)arylene,

R⁹ is C₂ to C₁₂ alkylene or C₅-C₁₂ (hetero)arylene,each R³ is independently H, —OH, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,preferably H or C₁-C₄ alkyl,R⁴ is H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or —N(R³)₂, preferably H, orC₁-C₄ alkyl.

The resulting polymer will have pendent guanidinyl groups of theformula:

whereinR¹ is H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,R² is a covalent bond, a C₂ to C₁₂ alkylene, a C₅-C₁₂ (hetero)arylene,

R⁹ is C₂ to C₁₂ alkylene or C₅-C₁₂ (hetero)arylene,each R³ is independently H, —OH, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,preferably H or C₁-C₄ alkyl,R⁴ is H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or —N(R³)₂, preferably H, orC₁-C₄ alkyl.

More particularly, the pendent ligand groups will be of the formula:

R⁹ is C₂ to C₁₂ alkylene or C₅-C₁₂ (hetero)arylene,each R³ is independently H, —OH, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,preferably H or C₁-C₄ alkyl,R⁴ is H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or —N(R³)₂, preferably H, orC₁-C₄ alkyl.

The reaction may be illustrated as follows:-(M^(CO))_(w)-(M^(Hydrophil))_(x)-(M^(hydrophob))_(z)-→-(M^(Lig))_(y)-(M^(Hydrophil))_(x)--(M^(hydrophob))_(z)-(M^(CO))_(w*)-,where-(M^(CO))_(w) are carbonyl functional monomer units having “w”polymerized monomer units,-(M^(Hydrophil))_(x)- are hydrophilic monomer units having “x”polymerized monomer units,-(M^(hydrophob))_(z)- are hydrophobic monomer units having “z”polymerized monomer units(M^(Lig))_(y) are ligand functional monomer units having “y” polymerizedmonomer units,where y is less than or equal to w; i.e. all or a portion of thecarbonyl groups are functionalized by the ligand compound of Formula I.The w, x and z subscripts correspond to the weight ranges of themonomers used: w may comprise 10 to 100 wt. % of the monomer mixture, xmay comprise 0 to 90 wt. % of the monomer mixture, and z may comprise 0to 20 wt. % of the monomer mixture. “y” indicates the number of carbonylfunctional groups functionalized with the ligand groups, and w*indicates the number of unfunctionalized carbonyl groups.

Alternatively to functionalizing the carbonyl functional polymer with aligand compound of formula I, the ligand functional polymer may beprepared by polymerizing a monomer of the formula:

whereinR¹ is H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,R² is a covalent bond, a C₂ to C₁₂ alkylene, a C₅-C₁₂ (hetero)arylene,

R⁹ is C₂ to C₁₂ alkylene or C₅-C₁₂ (hetero)arylene,each R³ is independently H, —OH, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,preferably H or C₁-C₄ alkyl,R⁴ is H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or —N(R³)₂, preferably H, orC₁-C₄ alkyl.X is —O— or —NR³—R⁶ is a C₂ to C₁₂ alkylene, andR⁷ is H or CH₃.

The monomer of Formula VI may be copolymerized with the hydrophilicmonomers previously described.

Alternatively, the ligand functional polymer may be prepared bypolymerizing a monomer of Formula VII. The monomer of Formula VII may becopolymerized with the hydrophilic monomers previously described.

whereinR¹ is H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,R² is a covalent bond, a C₂ to C₁₂ alkylene, a C₅-C₁₂ (hetero)arylene,

R⁹ is C₂ to C₁₂ alkylene or C₅-C₁₂ (hetero)arylene,each R³ is independently H, —OH, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,preferably H or C₁-C₄ alkyl,R⁴ is H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or —N(R³)₂, preferably H, orC₁-C₄ alkyl, and R⁷ is H or CH₃.

The disclosure further provides a functionalized substrate comprising abase substrate and a grafted or ungrafted coating of the ligandfunctionalized polymer thereon. Preferably the base substrate is aporous base substrate having interstitial and outer surfaces.

The base substrate may be formed from any suitable metallic,thermoplastic, or thermoset material. The material may be an organic orinorganic polymeric material. Suitable organic polymeric materialsinclude, but are not limited to, poly(meth)acrylates,poly(meth)acrylamides, polyolefins, poly(isoprenes), poly(butadienes),fluorinated polymers, chlorinated polymers, polyamides, polyimides,polyethers, poly(ether sulfones), poly(sulfones), poly(vinyl acetates),copolymers of vinyl acetate, such as poly(ethylene)-co-poly(vinylalcohol), poly(phosphazenes), poly(vinyl esters), poly(vinyl ethers),poly(vinyl alcohols), and poly(carbonates). Suitable inorganic polymericmaterials include, but are not limited to, quartz, silica, glass,diatomaceous earth, and ceramic materials.

Suitable polyolefins include, but are not limited to, poly(ethylene),poly(propylene), poly(1-butene), copolymers of ethylene and propylene,alpha olefin copolymers (such as copolymers of ethylene or propylenewith 1-butene, 1-hexene, 1-octene, and 1-decene),poly(ethylene-co-1-butene) and poly(ethylene-co-1-butene-co-1-hexene).

Suitable fluorinated polymers include, but are not limited to,poly(vinyl fluoride), poly(vinylidene fluoride), copolymers ofvinylidene fluoride (such as poly(vinylidenefluoride-co-hexafluoropropylene), and copolymers ofchlorotrifluoroethylene (such aspoly(ethylene-co-chlorotrifluoroethylene).

Suitable polyamides include, but are not limited to,poly(iminoadipoyliminohexamethylene),poly(iminoadipoyliminodecamethylene), and polycaprolactam. Suitablepolyimides include, but are not limited to, poly(pyromellitimide).

Suitable poly(ether sulfones) include, but are not limited to,poly(diphenylether sulfone) and poly(diphenylsulfone-co-diphenyleneoxide sulfone).

Suitable copolymers of vinyl acetate include, but are not limited to,poly(ethylene-co-vinyl acetate) and such copolymers in which at leastsome of the acetate groups have been hydrolyzed to afford variouspoly(vinyl alcohols).

The base substrate may be in any form such as particles, fibers, filmsor sheets. Suitable particles include, but are not limited to, magneticparticles, organic particles, inorganic particles, and porous andnonporous particles. Preferably the base substrate is porous. Suitableporous base substrates include, but are not limited to, porousparticles, porous membranes, porous nonwoven webs, and porous fibers.

In some embodiments, the porous base substrate is formed from propylenehomo- or copolymers, most preferably propylene homopolymers.Polypropylene polymers are often a material of choice for porousarticles, such as nonwovens and microporous films, due to propertiessuch as non-toxicity, inertness, low cost, and the ease with which itcan be extruded, molded, and formed into articles.

In many embodiments, the porous base substrate has an average pore sizethat is typically greater than about 0.2 micrometers in order tominimize size exclusion separations, minimize diffusion constraints andmaximize surface area and separation based on binding of a targetmolecule. Generally, the pore size is in the range of 0.1 to 10micrometers, preferably 0.5 to 3 micrometers and most preferably 0.8 to2 micrometers when used for binding of viruses. The efficiency ofbinding other target molecules may confer different optimal ranges.

Suitable porous base substrates include, but are not limited to, porousand microporous membranes, particles, nonwoven webs, and fibers. In someembodiments, the porous base substrate is a microporous membrane such asa thermally-induced phase separation (TIPS) membrane. TIPS membranes areoften prepared by forming a homogenous solution of a thermoplasticmaterial and a second material above the melting point of thethermoplastic material. Upon cooling, the thermoplastic materialcrystallizes and phase separates from the second material. Thecrystallized thermoplastic material is often stretched. The secondmaterial is optionally removed either before or after stretching.Microporous membrane are further disclosed in U.S. Pat. Nos. 4,539,256(Shipman), 4,726,989 (Mrozinski), 4,867,881 (Kinzer), 5,120,594(Mrozinski), 5,260,360 (Mrozinski et al.), and 5,962,544 (Waller), allof which are assigned to 3M Company (St. Paul, Minn.). Further, themicroporous film can be prepared from ethylene-vinyl alcohol copolymersas described in U.S. Pat. No. 5,962,544 (Waller).

Some exemplary TIPS membranes comprise poly(vinylidene fluoride) (PVDF),polyolefins such as polyethylene homo- or copolymers or polypropylenehomo- or copolymers, vinyl-containing polymers or copolymers such asethylene-vinyl alcohol copolymers and butadiene-containing polymers orcopolymers, and acrylate-containing polymers or copolymers. For someapplications, a TIPS membrane comprising PVDF is particularly desirable.TIPS membranes comprising PVDF are further described in U.S. Pat. No.7,338,692 (Smith et al.).

In another exemplary embodiment the porous bases substrate comprises anylon microporous film or sheet, such as those described in U.S. Pat.Nos. 6,056,529 (Meyering et al.), 6,267,916 (Meyering et al.), 6,413,070(Meyering et al.), 6,776,940 (Meyering et al.), 3,876,738 (Marinacchioet al.), 3,928,517, 4,707,265 (Knight et al.), and 5,458,782 (Hou etal.).

In other embodiments, the porous base substrate is a nonwoven web whichmay include nonwoven webs manufactured by any of the commonly knownprocesses for producing nonwoven webs. As used herein, the term“nonwoven web” refers to a fabric that has a structure of individualfibers or filaments which are randomly and/or unidirectionally interlaidin a mat-like fashion.

For example, the fibrous nonwoven web can be made by wet laid, carded,air laid, spunlaced, spunbonding or melt-blowing techniques orcombinations thereof. Spunbonded fibers are typically small diameterfibers that are formed by extruding molten thermoplastic polymer asfilaments from a plurality of fine, usually circular capillaries of aspinneret with the diameter of the extruded fibers being rapidlyreduced. Meltblown fibers are typically formed by extruding the moltenthermoplastic material through a plurality of fine, usually circular,die capillaries as molten threads or filaments into a high velocity,usually heated gas (e.g. air) stream which attenuates the filaments ofmolten thermoplastic material to reduce their diameter. Thereafter, themeltblown fibers are carried by the high velocity gas stream and aredeposited on a collecting surface to from a web of randomly disbursedmeltblown fibers. Any of the non-woven webs may be made from a singletype of fiber or two or more fibers that differ in the type ofthermoplastic polymer and/or thickness.

Further details on the manufacturing method of non-woven webs of thisinvention may be found in Wente, Superfine Thermoplastic Fibers, 48INDUS. ENG. CHEM. 1342 (1956), or in Wente et al., Manufacture OfSuperfine Organic Fibers, (Naval Research Laboratories Report No. 4364,1954).

In one embodiment the base substrate may have a coating of the ligandfunctional (co)polymer on a surface thereon. Useful coating techniquesinclude applying a solution or dispersion of the (co)polymer, optionallyincluding a crosslinker, onto the base substrate. Polymer application isgenerally followed by evaporating the solvent to form the polymercoating. Coating methods include the techniques commonly known as dip,spray, knife, bar, slot, slide, die, roll, or gravure coating. Coatingquality generally depends on mixture uniformity, the quality of thedeposited liquid layer, and the process used to dry or cure the liquidlayer.

In some embodiments, the carbonyl functional (co)polymer is first coatedon the base substrate and subsequently reacted with the ligand compoundof Formula I. Useful crosslinkers for the coating operation in theseinstances include carbonyl reactive compounds such as polyamines,polyhydrazines, and polyhydrazides.

In other embodiments, the ligand functional (co)polymer itself is coatedon the base substrate. Useful crosslinkers in these instances includeamine reactive compounds such as bis- and polyepoxides, polycarboxylicacids and their derivatives (e.g., acid chlorides), polyisocyanates, andformaldehyde-based crosslinkers such as hydroxymethyl and alkoxymethylfunctional crosslinkers, such as those derived from urea or melamine.

In other embodiments, the ligand functional copolymer is coated on thebase substrate by polyelectrolyte layer-by-layer coating techniques,such as those described in EP 472,990.

In another embodiment, the ligand-functional polymer may be grafted tothe surface of the base substrate; i.e. a covalent bond is formedbetween the ligand functional polymer and the polymer base substrate.The covalent bond may be formed by displacement, condensation or freeradical methods. The nature of the grafting depends on the type ofpolymer used for the base substrate.

In some embodiments, the base polymer has carbonyl-reactive functionalgroups, such as amines on the surface thereof. These surface functionalgroups may react with extant carbonyl functional groups on the ligandfunctional polymer. In another embodiment, the surface of the substratemay be provided with amine-reactive functional groups, such as halide,epoxy, ester, isocyanate groups, that can react with the guanidinogroups of the ligand functionalized polymer.

In some embodiments the polymer may be grafted to the surface of asubstrate by ionizing radiation-initiated graft polymerization of amonomer such as those of Formulas VI or VII, optionally with otherhydrophilic or hydrophobic monomers previously described. Alternatively,a carbonyl functional monomer may be grafted to the surface of asubstrate by ionizing radiation-initiated graft polymerization, followedby functionalization by reaction with a ligand compound of Formula I.

In some embodiments, the surface of the substrate may be free radicallyfunctionalized with a grafting monomer having a free-radicallypolymerizable group and a second functional group reactive with theligand functional polymer. Such monomers may include isocyanatoethyl(meth)acrylate or glycidyl (meth)acrylate.

The grafting monomers can graft (i.e. forming a covalent bond) to thesurface of the base substrate when exposed to an ionizing radiationpreferably e-beam or gamma radiation. That is, reaction of(meth)acryloyl groups of the grafting monomers with the surface of theporous base substrate in the presence of the ionizing radiation resultsin the reaction of ethylenically unsaturated free-radicallypolymerizable groups being directly grafted to the base substrate viathe acrylate group, and further provides the surface of the substratewith a reactive functional groups that may be subsequently reacted withthe ligand functional polymer.

The methods of the present disclosure involve the irradiation of porousor non-porous substrate surfaces with ionizing radiation to prepare freeradical reaction sites on such surfaces upon which the functionalmonomers are grafted. The functional groups of the functional monomersthen allow the ligand functional polymer to be grafted to the surface ofthe substrate. “Ionizing radiation” means radiation of a sufficientdosage and energy to cause the formation of free radical reaction siteson the surface(s) of the base substrate. Ionizing radiation may includebeta, gamma, electron-beam, x-ray and other electromagnetic radiation.In some instances, corona radiation can be sufficiently high energyradiation. The radiation is sufficiently high energy, that when absorbedby the surfaces of the base substrate, sufficient energy is transferredto that support to result in the cleavage of chemical bonds in thatsupport and the resultant formation of a free radical site on thesupport.

The method of grafting (or coating) a ligand functionalized polymer tothe surface of the substrate alters the original nature of the basesubstrate, as the substrate bears a grafted or ungrafted coating of theligand functional polymer. The present invention enables the formationof ligand functionalized polymer substrates having many of theadvantages of a base substrate (e.g., mechanical and thermal stability,porosity), but with enhanced affinity for biological species such asviruses, resulting from the monomers and steps used to form a givenfunctionalized substrate.

The porous substrates having a coating of ligand-functionalized polymerare particularly suited as filter media, for the selective binding andremoval of contaminating proteins, cells, cell debris, microbes, nucleicacids, and/or viruses from biological samples. The present disclosurefurther provides a method for the removal of target biological speciesfrom a biological sample by contacting the sample with the ligandpolymer functionalized substrate as described herein.

The ligand functionalized (co)polymer (either the polymer per se, or asubstrate having a coating thereof) is useful for the purification ofbiological or other fluid samples comprising biologically derivedspecies (biological species). Biological species include, but are notlimited to, cells, cell debris, proteins, nucleic acids, endotoxins, andviruses. Cells and cell debris include those derived from archaea,bacteria, and eucaryota. Bacteria include, but are not limited to,Gram-negatives such as Pseudomonas species, Escherichia coli,Helicobacter pylori, and Serratia marcesens; Gram-positives such asStaphylococcus species, Enterococcus species, Clostridium species,Bacillus species, and Lactobacillus species; bacteria that do not staintraditionally by Gram's method such as Mycobacterium species, andnon-vegetative forms of bacteria such as spores. Eucaryota include, butare not limited to, animal cells, algae, hybridoma cells, stem cells,cancer cells, plant cells, fungal hyphae, fungal spores, yeast cells,parasites, parasitic oocysts, insect cells, and helminthes. Proteins,include, but are not limited to, natural proteins, recombinant proteins,enzymes, and host cell proteins. Viruses include, but are not limitedto, enveloped species such as Herpesviruses, Poxviruses, Adenoviruses,Papovaviruses, Coronaviruses, retroviruses such as HIV, andPlasmaviridae; and non-enveloped species such as Caliciviridae,Corticoviridae, Myoviridae, and Picornaviridae.

In some embodiments, the biological species being removed from the fluidis the object of the purification. For example, a recombinant protein orenzyme may be prepared in cell culture, the (co)polymer can be added toflocculate the protein or enzyme, and the precipitate can be separatedas the first step in the purification process for the protein or enzyme.In another example, the (co)polymer or a substrate with a coatingthereof, may be used to capture microorganisms from a fluid as the firststep in a process of concentrating, enumerating, and/or identifying themicroorganisms.

In other embodiments, the biological species being removed from thefluid is a contaminant that must be removed prior to additionalprocessing steps for the fluid. The polymer can be used as a flocculantto facilitate the removal of cells and cell debris from a cell cultureor fermentation broth prior to, subsequent to, or in place of acentrifuge or depth filtration operation. For example, the (co)polymercan be used to flocculate cells in a cell culture broth prior tocentrifugation, and thereby improve the efficiency with which thecentrifugation process separates the cell mass from the liquid centrate.Alternatively, it can be added to the liquid centrate after acentrifugation step to flocculate suspended cell debris and dissolvedhost cell proteins and nucleic acids, thereby increasing the efficiencyof a subsequent depth filtration step. It can be used to flocculate orprecipitate suspended bacteria, viruses, or other microorganisms. It canbe used to precipitate either desired or contaminating proteins ornucleic acids from solution. Significantly, the ligand functional(co)polymers, or substrates having a coating thereof, are useful underconditions of high salt concentration or high ionic strength, i.e., theyare “salt tolerant”. The term “salt” is meant to include all lowmolecular weight ionic species which contribute to the conductivity ofthe solution. The importance of utility of the ligand functional(co)polymers in the presence of salt is that many process solutions usedin biopharmaceutical or enzyme manufacture have conductivities in therange of 15-30 mS/cm (approximately 150-300 mM salt) or more. Salttolerance can be measured in comparison to that of the conventionalquaternary amine or Q ligand (e.g. trimethylammonium ligand), whoseprimarily electrostatic interactions with many biological speciesrapidly deteriorates at conductivities three- to six-fold less than thetarget range. For example, membranes derivatized with the conventional Qligand exhibit a drop in φX174 viral clearance from a six log-reductionvalue (LRV) to a one (1) LRV in going from 0 to 50 mM NaCl (ca. 5-6mS/cm conductivity). Viruses such as φX174 which have pIs close to 7(are neutral or near-neutral) are extremely difficult to remove fromprocess streams. Similar problems are observed when attempting to removeother biological species from process fluids. For example, whenattempting to remove positively charged proteins such as host cellproteins through the use of filtration devices functionalized withconventional Q ligands, the process fluid may have to be dilutedtwo-fold or more in order to reduce the conductivity to an acceptablerange. This is expensive and dramatically increases the overallprocessing time.

When used as a flocculant, the amount of ligand functional (co)polymerthat is added relative to the amount of sample can vary over a widerange. Generally, the amount added will produce a final concentration of(co)polymer in the mixture of from about 0.01 micrograms/mL to about5000 micrograms/mL. The optimal amount of (co)polymer added will dependupon the concentration of the species one desires to flocculate.Typically, the amount of polymer relative to the amount of species beingflocculated will be in the range of 0.01% to 100% by weight, preferably0.05%-30% by weight, more preferably about 0.1%-10% by weight. Theoptimal amount is readily assessed by challenging the sample with aseries of polymer concentrations as is well known in the art. While theabove concentration ranges are typical, one skilled in the art willrealize that other ranges may work in some instances. Flocculationefficiency also depends upon the physical and chemical characteristicsof the species being flocculated. For example, we have found thatoptimal flocculation of the near neutral virus φX174 from aqueoussuspension occurs at a polymer to virus weight ratio of about 800-1000%.

The biological sample is contacted with the ligand functionalizedpolymer (either the polymer per se, or a substrate having a coatingthereof) for a time sufficient to interact and form a complex with thetarget biological species disposed (dissolved or suspended) in thesolution when the solution comprises from 0 to about 50 mM salt,preferably when the solution comprises from 0 to about 150 mM salt, morepreferably when the solution comprises from 0 to about 300 mM salt orhigher, such that the concentration of the target biological speciesremaining disposed in the solution is less than 50% of its originalconcentration. It is more preferred that the solution is contacted withthe ligand functionalized polymer for a time sufficient to interact andform a complex with the target biological species disposed in thesolution when the solution comprises from 0 to about 50 mM salt,preferably when the solution comprises from 0 to about 150 mM salt, morepreferably when the solution comprises from 0 to about 300 mM salt orhigher, such that the concentration of the target biological speciesremaining disposed in the solution is less than 10% of its originalconcentration. It is still more preferred that the solution is contactedwith the ligand functionalized polymer for a time sufficient to interactand form a complex with the target biological species disposed in thesolution when the solution comprises from 0 to about 50 mM salt,preferably when the solution comprises from 0 to about 150 mM salt, morepreferably when the solution comprises from 0 to about 300 mM salt orhigher, such that the concentration of the target biological speciesremaining disposed in the solution is less than 1% of its originalconcentration.

In many embodiments the ligand functionalized (co)polymer, beingpositively charged in aqueous media, will bind near neutral ornegatively charged species to the ligand functional group of Formula IIwhile other species (e.g., positively charged proteins such asmonoclonal antibodies) will be excluded or repelled from the ligandfunctionalized substrate. In addition, as previously described, thesubstrate may be directly or indirectly grafted with one or more ionicmonomers. In particular, the ligand functionalized polymer may comprisegrafted ionic groups that are positively charged at the selected pH ofthe biological sample solution to enhance electrostatic charge repulsionof proteins, such as monoclonal antibodies, many of which are chargedpositive at neutral pH, and ligand functional groups of Formula II toprovide salt tolerance.

In some embodiments the ligand functionalized polymer and coatedsubstrate containing the bound biological species are disposable. Insuch embodiments, the binding of the biological species to the ligandfunctionalized polymer is preferably essentially irreversible becausethere is no need to recover the bound biological species. Nonetheless,if desired, one can reverse the binding of biological species byincreasing the ionic strength or changing the pH of an eluting solution.

The substrate, having a grafted or ungrafted coating of the ligandfunctionalized polymer may be any previously described, but ispreferably a microporous membrane. The membrane pore size desired isfrom 0.1 to 10 μm, preferably 0.5 to 3 micrometers and most preferably0.8 to 2 micrometers. A membrane with a high surface area for theinternal pore structure is desired, which typically corresponds to finepore sizes. However, if the pore size is too small, then the membranetends to plug with fine particulates present in the sample solution.

If desired, efficiency of binding and capture may be improved by using aplurality of stacked, ligand functionalized polymer coated porousmembranes as a filter element. Thus the present disclosure provides afilter element comprising one or more layers of the porous, ligandfunctionalized polymer coated substrate. The individual layers may bethe same or different, and may have layers of different porosity, anddegree of grafting by the aforementioned grafting monomers. The filterelement may further comprise an upstream prefilter layer and downstreamsupport layer. The individual filter elements may be planar or pleatedas desired.

Examples of suitable prefilter and support layer materials include anysuitable porous membranes of polypropylene, polyester, polyamide,resin-bonded or binder-free fibers (e.g., glass fibers), and othersynthetics (woven and non-woven fleece structures); sintered materialssuch as polyolefins, metals, and ceramics; yarns; special filter papers(e.g., mixtures of fibers, cellulose, polyolefins, and binders); polymermembranes; and others.

In another embodiment, there is provided a filter cartridge includingthe above-described filter element. In yet another embodiment, there isprovided a filter assembly comprising the filter elements and a filterhousing. In a further embodiment, this invention relates to a method ofcapture or removal of a target biological species comprising the stepsof:

a) providing the filter element comprising one of more layers of theligand functionalized base substrate of this disclosure, and

b) allowing a moving biological solution containing a target biologicalspecies to impinge upon the upstream surface of the filter element for atime sufficient to effect binding of a target biological species.

The present invention is described above and further illustrated belowby way of examples, which are not to be construed in any way as imposinglimitations upon the scope of the invention. On the contrary, it is tobe clearly understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which, after readingthe description herein, may suggest themselves to those skilled in theart without departing from the spirit of the present invention and/orthe scope of the appended claims.

EXAMPLES Examples 1-5

Diacetone acrylamide (40 grams), ethanol (60 grams) and VAZO 67 (0.2gram) were charged to an 8 ounce glass bottle. The mixture was purgedwith a slow stream of nitrogen gas for 5 minutes, sealed, and thentumbled in a water bath equilibrated to 55° C. for 24 hours to convertthe monomer to polymer. A portion of this polymer solution (4.07 grams)was diluted with methanol (16.48 grams) and mixed with aminoguanidinehydrochloride (1.08 grams, TCI America, Portland, Oreg.).Trifluoroacetic acid (4 drops) was added as catalyst, and the solutionwas mixed for 2 hours at ambient temperature. IR and ¹H-NMR analysesconfirmed the formation of poly(diacetoneacrylamide guanylhydrazone)(polymer 1).

By similar procedures, polymers 2-5 were prepared by converting 20, 40,60, and 80% of the repeat units to guanylhydrazone functionality.

Examples 6-10

Copolymers of dimethylacrylamide and diacetoneacrylamide were preparedin methanol solution by the procedure described in Example 1. Thecopolymers, containing 20, 40, 60, 80 and 0% by weightdimethylacrylamide, were reacted with aminoguanidine by the proceduredescribed in Example 1 except that concentrated hydrochloric acid wasused as catalyst instead of trifluoroacetic acid, to produce thecorresponding guanylhydrazone copolymers 6-10.

Example 11

Guanylhydrazone polymers 1-5 were assayed for BSA precipitationaccording to the following procedure. Results are shown in FIG. 1.

A solution of bovine serum albumin (BSA, Sigma-Aldrich) was prepared in10 mM MOPS buffer (4-morpholinepropanesulfonic acid), pH 7.5, anddetermined to have a concentration of BSA of 4.0 mg/mL. A series of BSAsolutions were prepared containing various concentrations of sodiumchloride according to the following Table 1:

TABLE 1 [NaCl] BSA 5M MOPS (mM, solution NaCl buffer final) (mL) (μL)(μL) 0 10 0 500 50 10 100 400 100 10 200 300 150 10 300 200 200 10 400100 250 10 500 0

Solutions of the polymers from Examples 1-5 were diluted with deionizedwater to 1% solids by weight and adjusted to pH 7.

A 5 mL polypropylene centrifuge tube was charged with 2.0 mL of BSAsolution, followed by 125 μL of diluted polymer solution. The centrifugetube was sealed and tumbled end over end for 30 minutes, thencentrifuged at 2000 rcf for 10 minutes. A BSA standard solution wasprepared by mixing 2 mL of original BSA solution with 125 μL of MOPSbuffer. A serial 1:1 dilution was performed to provide a total of 7 BSAstandards. These seven standards were pipetted (200 μL) in triplicateinto wells of a 96-well microtitration plate, along with triplicatesamples of the supernates from each polymeric flocculant beingevaluated. Three wells containing DI water as a blank were alsoincluded. The plate was analyzed using a SpectraMAX 250 MicroplateSpectrophotometer System (Molecular Devices Corp, Sunnyvale, Calif.)using a wavelength of 293 nm. Comparison of the absorptions of theflocculant solutions to those of the standards provided a measure of thepercentage of BSA precipitated.

Comparative Example 1 Poly(MethacrylamidopropyltrimethylammoniumChloride) (pMAPTAC)

MAPTAC (160 grams of a 50% by weight solution in water, from Aldrich,Milwaukee, Wis.), ethanol (40 grams) and sodium persulfate (0.4 gram)were charged to a 16 ounce glass bottle. The mixture was purged with aslow stream of nitrogen gas for 10 minutes, sealed, and then tumbled ina water bath equilibrated to 55° C. for 24 hours to convert the monomerto polymer. This polymer solution was diluted with deionized water (80grams) and ethanol (40 grams) and mixed well. A sample for evaluation asa flocculant was prepared by dilution of a portion of this polymer to 1%solids by weight with deionized water, pH 7. A BSA precipitation testwas conducted as described in Example 11, and the pMAPTAC was found tobe a good flocculator at 0 mM NaCl, but it left 42%, 73%, and 100% ofthe BSA in solution at 50 mM, 100 mM, and 150 mM NaCl, respectively.

Example 12 Virus Flocculation

Aqueous suspensions of φX174 bacteriophage (ca. 10⁹ pfu/mL) wereprepared in 10 mM TRIS((hydroxymethyl)aminomethane) pH 8.0 containing 0,50 mM, and 150 mM NaCl. Aqueous solutions of flocculator polymers wereprepared in DI water, pH 7, at 0.001% polymer by weight. 16 μL ofpolymer solution were added to a 2 mL sample of bacteriophage suspensionin a centrifuge tube. The tube was sealed, vortexed, and rotatedend-over-end for 2 hours. The tubes were then centrifuged at 3000 rcffor 10 minutes, and the resultant suspensions were filtered through a0.45 micron sterile syringe filter (GHP Acrodisc, Pall Life Sciences). A10-fold dilution series was prepared.

One mL of each dilution was mixed with 1 mL E. coli culture (grown to anoptical density of 0.3-0.6 when measured at 550 nm). After waiting 5minutes, a sterile pipet was used to mix 4.5 mL TSA Top agar with thedilution/E. coli mixture and plated on TSB plates. After the top agarhad solidified, the plates were inverted and placed in a 37° C.incubator overnight. Plates were then removed from the incubator andφX174 plaques were counted and recorded. A dilution series of theoriginal virus suspension was also evaluated in a similar manner.Comparison of the results allowed estimation of the LRV (log reductionin viral load) as a result of the flocculant treatment. Results forseveral polymers are listed in the following Table 2:

TABLE 2 φX174 Polymer LRV Example 1 (0 NaCl) 4.9 Example 1 (50 mM NaCl)2.5 Example 1 (150 mM NaCl) 2.9 Comparative Example 1 6.3 (0 NaCl)Comparative Example 1 0.1 (50 mM NaCl) Comparative Example 1 0.5 (150 mMNaCl)

Example 13

Poly(vinylmethylketone) (1.0 gram, from Scientific Polymer Products,Ontario, New York) was dissolved in 4 grams of ethyl acetate. To thissolution was added methanol (5 grams), aminoguanidine hydrochloride(1.58 grams), and concentrated hydrochloric acid (1 drop). The mixturewas mixed for 1 hour, followed by the addition of methanol (5 grams) anddeionized water (5 grams) to produce an orange-colored solution.Infrared and NMR analysis confirmed the conversion to theguanylhydrazone polymer. The polymer was diluted to 1% solids by weightwith deionized water, pH 7. for evaluation as a BSA precipitant. Thispolymer showed >50% BSA precipitation up to 250 mM NaCl concentration.

Comparative Example 2 Poly(diallyldimethylammonium chloride) (pDADMAC)

To a 1 liter, 3-necked round bottom flask equipped with an overheadstirrer, condenser, heating mantle, thermocouple, and nitrogen inlet wascharged DADMAC (105.6 grams of a 65% solids aqueous solution, fromAldrich, Milwaukee, Wis.), tetrasodium ethylenediaminetetraacetic acid(6 μL of a 20% solution by weight in deionized water), deionized water(121.8 grams), and 2,2′-azobis(2-amidinopropane) dihydrochloride (1.74grams). Stirring was begun and the mixture purged with a slow stream ofnitrogen gas for 15 minutes. The temperature of the stirring mixture wasthen slowly raised to 60° C. over one hour, then maintained at thattemperature for an additional 24 hours. NMR analysis confirmed theformation of the expected polymer.

Examples 14-16

Copolymers of DADMAC with diacetoneacrylamide by procedures similar tothat described in Comparative Example 2. The copolymers, containing 20,40, and 80% by weight diacetoneacrylamide, were reacted withaminoguanidine by the procedure described in Example 1, to produce thecorresponding 20, 40, and 80% guanylhydrazone copolymers 14-16,respectively. Solutions of these copolymers and of pDADMAC were preparedat 1% by weight in deionized water (pH 7) and evaluated in the BSAprecipitation test. These flocculants displayed 0%, 30%, 62%, and 92%BSA precipitation at 100 mM NaCl for Comparative Example 2, Ex. 14, Ex.15, and Ex. 16, respectively.

Example 17

A Geobacillus stearothermophilus purified spore suspension was providedby 3M consisting of approximately 1.4% by weight cell debris and spores.Test samples of broth containing 0, 100, 200, and 300 mM NaCl wereprepared by a procedure similar to that described in Example 11.Solutions of polymers were prepared at 1.0% solids in DI water, pH 7,from the modified polymers of Examples 1, 6, and 8. A 1:4 dilutionseries of each of these polymers was prepared to provide a total of 6polymer concentrations. Then 2 mL of broth sample was mixed with 0.5 mLof polymer solution, and the mixture was tumbled for 30 minutes, thencentrifuged at 200 rcf for 5 minutes. Standards were prepared by mixing2 mL of broth with 0.5 mL of DI water, carrying the mixture through thesame mixing/centrifugation procedure, then preparing a 2-fold serialdilution (6 samples) from the supernate. Supernatants from the testsolutions and from the standards were pipetted into a 96-wellmicrotitration plate and assayed by absorbance measurement at 650 nm.Comparison of the absorptions of the flocculant solutions to those ofthe standards provided a measure of the flocculation efficiencies.Results are presented in the following Table 3:

TABLE 3 % Turbidity Removed 100 mM 200 mM 300 mM Polymer (w/v %) (μg/ml)0 mM NaCl NaCl NaCl NaCl Example 1 0.2 2000 54.7 95.9 98.9 98.7 ″ 0.05500 99.5 97.8 98.1 98.6 ″ 0.0125 125 95.7 97.0 95.0 92.1 ″ 0.00312531.25 82.2 83.9 81.5 78.6 ″ 0.000781 7.81 76.6 73.5 71.5 68.0 ″ 0.0001951.95 34 22.4 17.9 15.3 Example 6 0.2 2000 0.0 0.0 0.0 0.0 ″ 0.05 500101.3 45.5 21.9 24.8 ″ 0.0125 125 96.8 97.7 97.8 98.5 ″ 0.003125 31.2583.5 82.1 81.6 78.5 ″ 0.000781 7.81 70.9 76.5 66.4 52.2 ″ 0.000195 1.9525.8 28 24.4 17.5 Example 8 0.2 2000 0.0 0.0 0.0 0.0 ″ 0.05 500 53.442.2 9.8 10.5 ″ 0.0125 125 90.0 74.2 14.6 37.9 ″ 0.003125 31.25 98.688.5 58.7 55.1 ″ 0.000781 7.81 80.0 75.1 67.7 56.3 ″ 0.000195 1.95 70.468.2 64.5 40.8

Examples 18-20 and Comparative Examples 3-4

A 20:80 w/w copolymer of N,N-dimethylaminopropylacrylamide (DMAPAm) anddiacetoneacrylamide (Comparative Example 3) was prepared in methanolsolution by the procedure described in Example 1.

A sample of this polymer solution was diluted to 20% solids by addingmore methanol, and enough butyl bromide was added to alkylate 50% of theDMAPAm units of the copolymer (Comparative Example 4).

Samples of Comparative Example 3 polymer solution were reacted withaminoguanidine hydrochloride by a procedure similar to that of Example 6to produce copolymers in which 50% and 100% of the diacetoneacrylamideunits were converted to guanylhydrazone units, respectively (Examples 18and 19).

A sample of Comparative Example 4 was reacted with aminoguanidinehydrochloride by a procedure similar to that of Example 6 to convert allof the diacetoneacrylamide units to guanylhydrazone units (Example 20).

Samples of each of the above examples were diluted to 1% solids withdeionized water, adjusted to pH 7, and evaluated for BSA precipitation(Table 4)

TABLE 4 [NaCl] % BSA Precipitated (mM) Comp. 3 Comp. 4 Ex. 18 Ex. 19 Ex.20 50 0 0 62 89 93 150 0 0 28 47 86 250 0 1 14 18 75

Similar copolymers were prepared by alkylation with dimethylsulfate,benzyl bromide and dodecylbromide, respectively, followed by conversionof the keto groups to guanylhydrazones. When tested for BSAprecipitation, a synergistic effect was again observed for alkylationplus guanylation.

Example 21

A 50:50 w/w copolymer of DADMAC with diacetoneacrylamide was prepared bya procedure similar to that described in Comparative Example 2. Asolution of this copolymer in methanol (1.32 grams of 30% solidssolution) was diluted to 2% solids with deionized water, mixed withdiaminoguanidine hydrochloride (74 milligrams) and concentratedhydrochloric acid (1 drop), and allowed to react at room temperatureovernight to form the bis-guanylhydrazone copolymer. The solution wasadjusted to pH 7 and diluted to 1% solids with DI water. This polymerexhibited good flocculation of BSA in solutions containing up to 250 mMsodium chloride.

Example 22

Diacetoneacrylamide (33.8 grams), methanol (400 mL), and aminoguanidinehydrochloride (22.1 grams) were placed in a 1 L round bottom flask andstirred magnetically until a homogeneous solution had formed.Trifluoroacetic acid (0.5 mL) was added and the mixture was stirredovernight at ambient temperature. The reaction mixture was concentratedon a rotary evaporator to produce a light yellow oil which crystallizedon standing. Diethyl ether (250 mL) was added and the crystalline masswas broken up, filtered, washed with additional ether, and dried undervacuum for 3 hours to give the corresponding guanylhydrazonehydrochloride monomer (53.5 grams, 95.7% yield). This monomer (10 grams)was mixed with deionized water (23 grams), methanol (7 grams), and2,2′-azobis(2-imidazolin-2-yl)propane (0.2 grams, from WAKO, Osaka,Japan). The mixture was purged with nitrogen gas for 15 minutes, thenpolymerized at 60° C. for 24 hours as described in Example 1. Thispolymer displayed flocculation properties identical to those of Example1.

Example 23

A polypropylene spunbond/melt blown/spunbond (SMS) nonwoven (50 gsmbasis weight) was coated with the polymer of Example 1 in alayer-by-layer process. Three coating baths were prepared:

-   -   Coating Bath #1: 1% wt/wt polyethylenimine (70,000 M_(n)) in        isopropyl alcohol;    -   Coating Bath #2: 0.5% wt/wt        poly(2-acrylamido-2-methyl-1-propanesulfonic acid, sodium salt)        in deionized water;    -   Coating Bath #3: 1% wt/wt polymer of Example 1 in deionized        water.        The following coating sequence was conducted (5 minute residence        time in each bath) with a 5 minute rinse with deionized water        after each coating layer was applied: #1, #2, #1, #2, #3, #2,        #3, #2, and #3. After drying at room temperature overnight, a 16        mm diameter disk was cut from the coated web. This disk was        placed in a 5 mL centrifuge tube, 2 mL of a Clostridium        sporogenes spore suspension (with and without added sodium        chloride) was added, and the mixture tumbled at room temperature        for 30 minutes. The UV absorbance of the supernatant solution        was measured at 640 nm and compared to the absorbance prior to        contact with the disk, and indicated 46% and 58% removal of        spores at 0 and 250 mM sodium chloride concentrations,        respectively.

Example 24

Aminosilane coated glass microscope slides (obtained from NewcomerSupply, Middleton, Wis.) were dip coated, in sequence, with coatingsolution #2 from Example 23, rinsed with deionized water, coatingsolution #3 from Example 23, rinsed with deionized water, and dried atroom temperature. The coated slides could then be used to capturebacteria or spores from aqueous media for enumeration or foridentification. A control aminosilane slide coated with only coatingsolution #2 was found to bind almost no bacteria or spores.

Examples 25-28

A diacetoneacrylamide solution was prepared at 10% by weight inisopropanol. Several sheet substrates (ca. 4 inches by 6 inches), bothmembrane and non-woven, as listed in the table below, were placed inpolyethylene bags (Ziploc™, S C Johnson, Racine, Wis.). Using adisposable pipette, the substrates were completely wetted with thediacetoneacrylamide solution. The excess solution was removed by zippingthe bags shut and running over the bags with a rubber roller. The pooledexcess solutions were removed with a paper towel. The bags were thenvacuum-packed into Foodsaver™ bags. The bags were deliberately notpurged of oxygen so as to minimize solvent evaporation. The samples wereplaced in an aluminum tote and exposed for 1 hour to Co-60γ radiation.The dose exposure corresponded to 5 kGy. The samples were taken out ofthe bags and washed with copious amounts of water to remove any tracesof homopolymer. The completely washed samples were then air dried.

The diacetoneacrylamide grafted membranes were then converted toguanylhydrazone functional membranes. An aminoguanidine solution wasprepared by dissolving 65 grams in 1 liter of methanol, and addingtrifluoroacetic acid (2.25 mL). Each membrane was placed in a 500 mLpolyethylene bottle, 100 mL of the aminoguanidine solution was added,the bottles were sealed, and the mixtures were placed on a roller atroom temperature overnight. Excess solution was removed, and themembranes were washed extensively with deionized water and dried.

24 mm disks were punched out of the sheets and placed in a centrifugetube. Bovine serum albumin solution (BSA, Sigma Aldrich) was prepared toa concentration of 0.75 mg/ml in 25 mM TRIS buffer, pH 8.0(tris(hydroxymethyl)aminomethane, Sigma). 4.5 ml of the BSA solution waspipetted into each centrifuge tube, the tubes were capped, and the tubeswere tumbled overnight. The supernatant solutions were analyzed by aUV-VIS spectrometer at 279 nm with background correction applied at 325nm. Static binding capacities for the samples were found to be between 1and 4 mg/mL. Analyses of the ungrafted substrates displayed bindingcapacities of <0.2 mg/mL.

TABLE Substrate Example 25 Polyvinylidene fluoride membrane, 2 μmnominal pore size Example 26 Nylon 6 melt-blown non-woven web Example 27Nylon 66 membrane, single reinforced layer nylon three zone membrane,nominal pore size 1.8 μm Example 28 Polypropylene non-woven web, sold as3M Doodleduster ™ cloth

Example 29

A 60 gsm nylon 6 meltblown nonwoven substrate with a 4.3 um effectivefiber diameter and sample size of about 30 cm×43 cm (1290 cm² or 200square inches) was purged of air and inserted into Ziploc bags in aglove box under nitrogen atmosphere (less than 20 ppm oxygen) andsealed. The Ziploc bag was then removed from the glove box, andirradiated to a dose level of 60 kGy (kilogray) by passing it throughthe electron beam. The bag was returned to the N₂ atmosphere-controlledglove box.

The Ziplock bag was opened and the nonwoven substrate was imbibed bypouring 150 grams of a nitrogen purged imbibing solution comprising; 17%diacetone acrylamide monomer, 10% polyethylene glycol with hydroxy endgroups, average molecular weight 4,600 g/mole (PEG 4600), 1.0% SartomerSR-550 PEG-acrylate and 72% water by weight inside the Ziploc bag withthe nonwoven substrate. The nonwoven is kept flat and is completely andevenly saturated with a slight excess of grafting solution. The Ziplocbag is sealed. The Ziploc bag remains inside of the nitrogen filledglove box to allow sufficient time for all of the grafting monomer tograft-polymerize by free radical reaction with the nonwoven substrate.After a minimum of 6 hrs or as long as about 18 hrs (overnight), thegrafted nylon nonwoven sample was removed from the glovebox and theZiploc bag and the grafted nonwoven was washed three times by soaking in2 liters of fresh DI water (to remove the PEG and any unreacted monomer)and then air dried to ambient conditions.

A solution of aminoguanidine (92 grams) and concentrated hydrochloricacid (3.45 mL) in deionized water (1500 mL total solution volume) wasprepared. A sheet of the grafted nonwoven (ca. 12 cm×40 cm) was placedin a 1 gallon plastic jar. A portion (500 mL) of aminoguanidine solutionwas added to the jar, which was then sealed and placed on a roller forovernight reaction at room temperature. The excess aminoguanidinesolution was decanted, the derivatized nonwoven sheet was rinsed under astream or deionized water for 15 minutes, and the sheet then allowed toair dry at room temperature. A 24 mm diameter disk was cut from thecoated web. This disk was placed in a 5 mL centrifuge tube, 4.5 mL of aBovine serum albumin (BSA) solution (ca. 3 mg/mL in 25 mM TRIS, pH 8.0)was added, and the mixture tumbled at room temperature overnight. The UVabsorbance of the supernatant solution was measured at 279 nm andcompared to the absorbance prior to contact with the disk to determinethe static binding capacity for BSA. The functionalized nonwoven wasfound to absorb 152 mg of BSA per gram of web.

Example 30

Using standard microbiological procedures, cultures of the followingwere prepared:

a) Bacillus atrophaeous (spores and cell debris)

b) Clostridium sporogenes (spores)

c) Escherichia coli (cells and cell debris)

d) Chinese hamster ovary (CHO) cells

e) Baker's yeast

When flocculation experiments were conducted similarly to thosedescribed in Example 17 on these mixtures, the ligand functionalpolymers of the invention consistently displayed good flocculatingability in the presence of sodium chloride concentrations in excess of50 mM.

Example 31

Aminoguanidine hydrochloride (1.11 grams), methanol (15 grams), andacetoacetoxyethyl methacrylate (2.2 grams) were stirred at roomtemperature and monitored periodically by NMR. After stirring for 21hours, NMR confirmed complete conversion of the methacrylate monomer tothe corresponding guanylhydrazone hydrochloride

1. A functionalized substrate comprising a porous base substrate and agrafted or ungrafted coating of a carbonyl-functional (co)polymerfunctionalized with pendent guanidinyl groups on the surface thereof,said pendent guanidinyl groups are of the formula:

wherein R¹ is H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl, R² is a covalentbond, a C₂ to C₁₂ alkylene, or a C₅-C₁₂ (hetero)arylene, a

R⁹ is C₂ to C₁₂ alkylene or C₅-C₁₂ (hetero)arylene, each R³ isindependently H, —OH, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl, each R⁴ isH, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or —N(R³)₂.
 2. The functionalizedsubstrate of claim 1 wherein the pendent guanidinyl groups are of theformula:

wherein R¹ is H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl, R⁹ is C₂ to C₁₂alkylene or C₅-C₁₂ (hetero)arylene, each R³ is independently H, —OH,C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl, each R⁴ is H, C₁-C₁₂ alkyl, C₅-C₁₂(hetero)aryl, or —N(R³)₂, preferably H, or C₁-C₄ alkyl, R⁵ is a divalentalkylene.
 3. The functionalized substrate of claim 1 wherein thecarbonyl-functional (co)polymer is selected from the group consisting ofcopolymers of; acrolein, vinyl methyl ketone, vinyl ethyl ketone, vinylisobutyl ketone, diacetone (meth)acrylamide, acetonyl acrylate, carbonmonoxide copolymer, and diacetone (meth)acrylate.
 4. The functionalizedsubstrate of claim 2 wherein the (co)polymer comprises polymerizedmonomer units of the formula:

VI wherein R¹ is H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl, R² is acovalent bond, a C₂ to C₁₂ alkylene, or a C₅-C₁₂ (hetero)arylene, a

R⁹ is C₂ to C₁₂ alkylene or C₅-C₁₂ (hetero)arylene, each R³ isindependently H, —OH, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl, R⁴ is H,C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or —N(R³)₂, X is —O— or —NR³— R⁶ is aC₂ to C₁₂ alkylene, and R⁷ is H or CH₃.
 5. The functionalized substrateof claim 2 where the (co)polymer comprises polymerized monomer units ofthe formula:

wherein R¹ is H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl, R² is a covalentbond, a C₂ to C₁₂ alkylene, or a C₅-C₁₂ (hetero)arylene,

R⁹ is C₂ to C₁₂ alkylene or C₅-C₁₂ (hetero)arylene, each R³ isindependently H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl, preferably H orC₁-C₄ alkyl, R⁴ is H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or —N(R³)₂,preferably H, or C₁-C₄ alkyl, and R⁷ is H or CH₃.
 6. The functionalizedsubstrate of claim 2 where the (co)polymer further comprises hydrophilicmonomer units.
 7. The functionalized substrate of claim 2 wherein the(co)polymer has the formula:-(M^(Lig))_(y)-(M^(Hydrophil))_(x)--(M^(hydrophob))_(z)-(M^(CO))_(w*)-,where -(M^(CO))_(w) are carbonyl functional monomer units having “w”polymerized monomer units, -(M^(Hydrophil))_(x)- are hydrophilic monomerunits having “x” polymerized monomer units, -(M^(hydrophob))_(z)- arehydrophobic monomer units having “z” polymerized monomer units(M^(Lig))_(y) are ligand functional monomer units having “y” polymerizedmonomer units, y is 10 to 100 wt. % of the monomer units; x is 0 to 90wt. % of the monomer units; and w* may be comprise 0 to 90 wt. % of themonomer units, and z is 0 to 20 wt. %, based on 100 wt. % totalmonomers.
 8. The functionalized substrate of claim 1 wherein theligand-functionalized polymer is coated on the porous substrate.
 9. Thefunctionalized substrate of claim 1 wherein the ligand-functionalizedpolymer is grafted on the substrate.
 10. The functionalized substrate ofclaim 1 wherein the porous base substrate is a microporous basesubstrate.
 11. The functionalized substrate of claim 1 wherein theporous base substrate is a nonwoven web.
 12. A method of separating abiological species from a fluid comprising contacting the fluid with thefunctionalized substrate of claim 1 whereby a complex comprising thefunctionalized substrate and the biological species is formed, andseparating the complex.
 13. The method of claim 12 wherein thebiological species are selected from cells, cell debris, viruses,proteins, nucleic acids, and endotoxins.
 14. The method of claim 12where the functionalized substrate is selected from a particle, a fiber,a film, or a sheet.
 15. The method of claim 12 where the functionalizedsubstrate is a woven or nonwoven web.